During the last several years our laboratory has studied three different dominantly-inherited autoinflammatory disorders. The first of these illnesses is the TNF receptor-associated periodic syndrome (TRAPS), which is characterized by oftentimes prolonged attacks of fever, serositis, migratory rash and myalgia, arthritis, periorbital edema, conjunctivitis, and, in some patients, systemic amyloidosis. In 1999 our group identified the first six mutations in the gene encoding the 55 kDa tumor necrosis factor receptor (TNFRSF1A) in seven families with dominantly-inherited recurrent fevers, and therefore proposed the name ?TRAPS? for this clinical condition. Initial mechanistic studies indicated a defect in the activation-induced shedding of the p55 (but not p75) TNF receptor, possibly leading to impaired homeostasis of the immune response. [unreadable] [unreadable] In 2002 we and a French group independently discovered dominantly-inherited de novo mutations in a second gene, CIAS1 (also known as NALP3 or PYPAF1), in about 50% of patients with a disorder known as neonatal onset multisystem inflammatory disease (NOMID) or chronic infantile neurologic cutaneous and articular (CINCA) syndrome. Manifestations of NOMID/CINCA may include daily fevers, an urticaria-like skin rash, chronic aseptic meningitis, uveitis, papilledema, sensorineural hearing loss, mental retardation, patellar and epiphyseal long bone overgrowth, and systemic amyloidosis. Two phenotypically milder conditions, familial cold autoinflammatory syndrome (FCAS) and Muckle-Wells syndrome (MWS), are caused by mutations in the same gene. CIAS1 encodes a protein, cryopyrin, that participates in a macromolecular complex called the inflammasome to regulate the activation of interleukin-1 (IL-1) beta.[unreadable] [unreadable] A third dominantly-inherited autoinflammatory disorder, denoted the syndrome of pyogenic arthritis with pyoderma gangrenosum and acne (PAPA), is caused by mutations in a protein known as proline serine threonine phosphatase interacting protein (PSTPIP1). PAPA is characterized by episodes of mono- or pauciarticular sterile pyogenic arthritis, which can be destructive if not treated, formation of open, purulent ulcers of the skin (pyoderma gangrenosum), and severe cystic acne. In 2003 our group discovered that PSTPIP1 binds pyrin, the protein mutated in familial Mediterranean fever (FMF), and that disease-associated mutations in PSTPIP1 lead to more avid binding to pyrin, and increased IL-1 beta activation, relative to healthy controls.[unreadable] [unreadable] Results of the Last Year[unreadable] [unreadable] Functional studies of mutant TNFRSF1A molecules: In collaboration with the laboratory of Dr. Richard Siegel, we have continued analyses of the functional consequences of TRAPS-associated TNFRSF1A mutations. In transfection systems, mutant receptors were found to have a number of functional abnormalities, including (1) impaired activation-induced ectodomain cleavage; (2) diminished binding to TNF; (3) lack of association with wild-type receptors through the pre-ligand assembly domain (PLAD); (4) impaired ability to signal apoptosis or NF kappa B activation; and (5) abnormal intracellular trafficking, with retention in the endoplasmic reticulum (ER). Current studies focus on the response of mouse embryonic fibroblasts from TRAPS knockin mice to ER stress. Studies of cytokine production by peripheral blood mononuclear cells from TRAPS patients show heightened production in response to low doses of LPS, again suggesting exaggerated inflammatory responses to subclinical stimuli. [unreadable] [unreadable] Mutational analysis in patients with clinical FCAS, MWS, or NOMID: In collaboration with Dr. Hal Hoffman, we screened new referrals for CIAS1 mutations. Of the 22 NOMID/CINCA patients, 5 had novel CIAS1 mutations (V262A, V351L, F443L, and 2 with L264F), 7 were carriers for previously described mutations, and 10 were mutation-negative. Of the 12 MWS patients, 1 had a novel mutation (F523C), 8 had previously-described mutations, and 3 were mutation-negative. Among the 18 FCAS patients, 3 were carriers for 2 novel mutations (C259W and Y563N), 13 were carriers for previously described mutations, and 2 were mutation-negative. Several mutation-negative patients were screened for mutations in 12 candidate genes chosen for similarity to CIAS1 or involvement in the caspase-1/IL-1 beta signaling pathway; no pathogenic changes were found. We also assessed the frequency of 3 known CIAS1 variants of uncertain significance, V198M, R488K, and Q703K, in the general population. Although the allele frequencies of the first two variants were less than 1%, suggesting that they may be reduced-penetrance mutations, the allele frequency of Q703K was 5%, indicating that it is unlikely to be a pathogenic change. Computational modeling of the significant disease-causing mutations suggested a structure in which the NACHT domains of 6 cryopyrin molecules form a hexagonal assembly, with nearly all of the mutations on a common surface.[unreadable] [unreadable] Studies of IL-1 inhibition in the cryopyrinopathies: In collaboration with Dr. Raphaela Goldbach-Mansky, we have conducted therapeutic trials of an FDA-approved recombinant IL-1 receptor antagonist, anakinra, in 18 patients with NOMID. The results are more thoroughly presented under project Z01 AR041138-04 OCD, but will be briefly summarized here. In the anakinra trial, a total of 18 NOMID patients were treated with anakinra; in 11, the drug was briefly withdrawn after 3 months until a flare occurred. Clinical and laboratory parameters improved dramatically while on study drug, withdrawal resulted in relapse within days, and retreatment led to rapid improvement. There were highly significant improvements in a disease-specific diary score at 3 and 6 months of treatment, as well as in the erythrocyte sedimentation rate, C-reactive protein, and serum amyloid A. There was also evidence of reduced inflammation of the central nervous system, including decreased headache, reduced opening pressure on lumbar puncture, and decreased cochlear and leptomeningeal enhancement on magnetic resonance imaging (MRI). Before treatment peripheral blood leukocytes from patients spontaneously produced high levels of IL-1 beta, and this decreased progressively with treatment. Gene expression profiling also revealed aberrantly elevated expression of IL-1 beta and genes downstream of IL-1 beta prior to treatment, which decreased on anakinra and returned with drug withdrawal. We are continuing the anakinra trial to study long-term safety and efficacy. We are also currently studying a longer-acting investigational soluble IL-1 receptor fusion protein in patients with FCAS/MWS.[unreadable] [unreadable] Generation of new animal models: We are currently in the process of developing new animal models for the cryopyrinopathies and PAPA syndrome. In collaboration with Dr. Hal Hoffman, we are developing models for FCAS (CIAS1 L3533P), MWS (A352V), and NOMID (D303N, Y570C). We are also generating PSTPIP1 knockout mice, and the PSTPIP1 E250Q PAPA knockin mouse.[unreadable] [unreadable] Conclusions and Significance[unreadable] [unreadable] Studies on the functional consequences of TNFRSF1A mutations suggest a more complex pathophysiologic model of TRAPS than previously appreciated, in which constitutive cellular activation may play a prominent role. Our data on CIAS1 mutations expand the mutational spectrum of the cryopyrinopathies, and suggest hypotheses for the structural implications of CIAS1 mutations. Therapeutic studies of IL-1 inhibitors in NOMID patients confirm the importance of cryopyrin in IL-1 regulation, and represent a major advance in the treatment of these patients. During the next year we will continue studies of the possible role of the ER stress response in TRAPS, continue therapeutic and molecular pathophysiologic studies of patients with cryopyrinopathies, and focus on new animal models as they become available.